1, and clinical scoring performed as described previously [28]. Samples for antibody,

viremia, and lymphocyte proliferation analyses were collected as indicated in Fig. 1, in dry, ethylene diamintetraacetic acid (EDTA), and heparinized tubes (BD Biosciences, USA), respectively. Viral RNA was extracted using a Magnatrix robot and a pan-BTV qPCR based on segment 1 (VP1) of BTV [29] was performed. The standard curve was obtained by dilution of a viral suspension (105.9 TCID50 equivalent units/ml), as performed previously [30]. The quantity of viral RNA is expressed in log10 TCID50 equivalent units/ml. ECE inoculation was performed as described previously [31], in five 12-day-old embryonated specific pathogen-free chicken eggs (Håtunaholm, Sweden) per calf blood sample collected on PID8. Dead embryos were scored as positive if they showed hemorrhages characteristic of BTV infection.

Protein Tyrosine Kinase inhibitor Embryos were homogenized after death or on day 7, after placement at +4 °C for at least 4 h. RNA was extracted from swabs of homogenized embryos and RT-qPCR performed as described above. Virus neutralizing assays were performed in duplicate on Vero cells, using serially diluted sera from 1:2 to 1:256 (as described previously [32]). BTV-specific CPE were identified under a light microscope after 5 days of incubation. The neutralizing titer was defined as the highest dilution GW-572016 purchase allowing neutralization of 100 TCID50 of BTV-8. Competitive (c) enzyme-linked immunosorbent assays (ELISAs) were used to measure specific serum antibodies to VP2 of BTV-8 and VP7 of any BTV serotype (ID Screen® Bluetongue Serotype 8 Competition and ID Screen® Bluetongue Competition, ID Vet, France, respectively), according to the manufacturer’s protocols. Results are expressed as 100% minus competition percentage (100 times [ODsample/ODnegative control]). Antibodies specific to NS1 and NS2 (BTV-2) were analyzed using indirect ELISAs as described previously [26]. Results are expressed as log10-transformed antibody titers, which were calculated by linear regression to the corrected OD (COD = ODprotein − ODbackground control) value of negative control sera at a

dilution factor of 10. For calculating means and performing statistical analysis, values under the detection threshold were set to that threshold (dilution factor 10). Peripheral blood mononuclear cells (PBMCs) were isolated 4-Aminobutyrate aminotransferase from heparinized blood of animals as previously described [33], then stored in liquid nitrogen. Cells were restimulated, in duplicate, as described previously [34], with 0.03–1 μg individual proteins (VP2, NS1, NS2) or 103.9 TCID50/well of UV-inactivated BTV-8 and relevant background controls (Sf9 cell lysate for VP2, NS1; non-transfected BL21-AI™ E. coli lysate for NS2; uninfected Vero cell lysate for virus). Absorbances were measured 7–16 h after addition of alamarBlue®-reagent (Invitrogen, UK), at 570 nm and 595 nm. OD (OD570nm − OD595nm) and COD values were calculated for all protein- and virus-specific stimulations.